The overall goals of this proposal are (1) investigate the structure and topology of the membrane-bound KCNE1 protein; (2) elucidate the binding mechanism of KCNE1 with the C-terminal domain of the KCNQ1 K+ channel; (3) identify structural/binding differences in disease-causing long QT syndrome (LQTS) E1 and Q1 mutations, and (4) develop new magnetic resonance techniques to study the structure of membrane proteins. The membrane-bound KCNE1 protein modulates the activity of the KCNQ1 voltage-gated K+ channel. KCNE1 is responsible for slowing the voltage-stimulated activation of KCNQ1 (IKs) and is essential for proper channel and heart function. Hereditary E1/Q1 mutations have been linked to LQTS, atrial fibrillation, sudden infant death syndrome, and deafness. A recently published solution NMR structure of KCNE1 in LMPG micelles reveals that KCNE1 adopts a unique curved alpha-helical secondary structure. Several structural biology studies have indicated that the structure of a protein in a micelle can change dramatically when the protein is embedded in a membrane. Recent CD data by the Lorigan lab on KCNE1 in proteoliposomes reveals dramatic changes in the secondary structure when compared to the micelle structure. We hypothesize that the structure of KCNE1 in a lipid bilayer differs from the solution NMR structure of KCNE1 in LMPG micelles. The three-dimensional structure of KCNQ1 or the KCNE1/KCNQ1 complex has not been determined. Furthermore, the structural nature of the binding interaction of E1 with Q1 is poorly understood and has only been investigated indirectly with biochemical binding and cross-linking assays. It is critical to study the structureof E1 with Q1 to properly describe the function and rhythm of a heartbeat. EPR spectroscopy will be used to directly probe the structural and dynamic properties of KCNE1 and the KCNE1/KCNQ1 complex. Transformative biophysical techniques will be developed to study the structural and dynamic properties of KCNE1 and the KCNE1/KCNQ1 complex in a membrane. These state-of-the-art pulsed EPR spectroscopic techniques will move the field forward by dramatically increasing sensitivity and distance measurements of membrane protein systems such as KCNE1. The following pertinent biological questions will be addressed in the specific aims: Which segments of KCNE1 are helical in a bilayer? Does KCNE1 have a curved or straight -helix in a lipid bilayer (which structural model is correct)? What is the structural topology of KCNE1 with respect to the membrane? How does KCNE1 bind to the cytoplasmic domain of the KCNQ1 K+ channel? Which proposed structural model is correct for the E1/Q1 complex? Do disease-causing E1 or Q1 LQTS mutations alter the structure or binding mechanism of the E1/Q1 complex?!